Urinary excretion of glycosaminoglycans in horses: Changes with age, training, and osteoarthritis

The aim of the current study was to investigate if the urinary glycosaminoglycans reflect the changes that occur in cartilages with age, training, and osteoarthritis in horses. The glycosaminoglycans were extracted by ion exchange chromatography from urine samples of 90 sedentary and athletic horses of different ages and genders, with or without osteoarthritis. The main urinary glycosaminoglycans in horses are chondroitin sulfate, dermatan sulfate, and heparan sulfate. A marked decrease occurred with age. Trace amounts of keratan sulfate were detected by immunochemical methods, and its concentration increased linearly with age. Athletes excreted fewer glycosaminoglycans in the urine than age-matched sedentary horses (including keratan sulfate), and sedentary horses with osteoarthritis excreted fewer total glycosaminoglycans, but more keratan sulfate, than their healthy counterparts. The chondroitin sulfate excreted in the urine by foals contained a low 6-sulfated disaccharide/4-sulfated disaccharide (Di6S/Di4S) ratio, as compared with adults and aged horses. Low Di6S/Di4S ratios were also found in the chondroitin sulfate excreted by healthy racehorses and by horses with osteoarthritis. Our data show clear correlations between urinary glycosaminoglycans in horses and age, physical exercise, and osteoarthritis. The combination of qualitative, quantitative, and structural analyses of these compounds may be useful for the diagnosis and follow-up of osteoarthritis in horses.

Introduction

Hyaline cartilage is a specialized connective tissue that resists and redistributes impact loading of the joint while providing a resilient articular surface. These properties depend on the hydration and structural organization of the large extracellular matrix molecules. The cartilage extracellular matrix is composed of a dense network of collagen fibers (typically types II, VI, IX, and XI) that entrap a high concentration of proteoglycans and small amounts of noncollagenous proteins. The main cartilage matrix proteoglycan is aggrecan, which corresponds to about 10% of the tissue dry weight. Aggrecan consists of a central core (∼200 kDa) with about 100 covalently linked chondroitin sulfate chains, 30 to 50 keratan sulfate chains, and O-linked and N-linked oligosaccharides. The core protein contains 3 globular domains (G1, G2, and G3) and a long extended domain between G2 and G3, to which the glycosaminoglycan chains are linked. Aggrecan forms macromolecular structures through stable interactions of G1 with hyaluronan. These aggrecan-hyaluronan interactions are stabilized by link protein, a glycoprotein with structural homology to G1.1, 2 Low-molecular-weight proteoglycans of the leucine-rich protein family—decorin, biglycan, and fibromodulin—have also been identified in the cartilage.³ Although these proteoglycans account for only a small proportion of the total glycosaminoglycans, they may be present in similar molar proportions to aggrecan.

We have previously shown that when growth and calcification processes occur in human cartilages, the ratio keratan sulfate/chondroitin sulfate is lower and the chondroitin sulfate is hybrid, containing both 4-sulfated and 6-sulfated disaccharide units.⁴ These changes were observed with age and also in osteoarthritis and cartilage tumors, both enchondromatosis and chondrosarcoma5, 6 Similar results were also reported by other authors.⁷

Proteoglycans are synthesized in the endoplasmic reticulum and Golgi apparatus, and their degradation occurs mostly in lysosomes, by the sequential action of proteases, glycosidases, deacetylases, and sulfatases. The majority of the proteoglycans are recycled, but small amounts of partially degraded glycosaminoglycans are excreted in the urine. Much of the information concerning the degradation of proteoglycans has been derived from the study of mucopolysaccharidoses, a group of genetic disorders characterized by accumulation in tissues and excretion in the urine of products that result of incomplete breakdown of proteoglycans, due to deficiency of 1 or more lysosomal hydrolases.⁸

Normal human urine contains small amounts of glycosaminoglycans (10–20 mg/L), and changes in urinary glycosaminoglycans have been recently reported in many diseases, such as renal lithiasis,⁹ interstitial cystitis,¹⁰ diabetes mellitus,¹¹ and chronic renal failure.¹² Although the determination of glycosaminoglycans in the urine is gradually gaining importance in the literature, the exact mechanism behind the urinary excretion of glycosaminoglycans is not completely understood.

It has been shown for different mammalian species, including rat,¹² dog, cat,¹³ and human,¹⁴ that the main urinary glycosaminoglycans are chondroitin sulfate, dermatan sulfate, and heparan sulfate. Trace amounts of keratan sulfate and hyaluronan have also been detected by immunochemical methods.15, 16

There are evidences suggesting that most of the urinary glycosaminoglycans have systemic origin and are filtered in the kidney. In the normal rat, cat, and human kidney, only trace amounts of dermatan sulfate were found, while chondroitin sulfate was not detected, indicating that the urinary chondroitin sulfate and dermatan sulfate do not come from the kidney.11, 13, 17 In contrast, chondroitin sulfate was the main glycosaminoglycan found in the plasma.¹³ Circulating glycosaminoglycans are thought to represent products of the metabolism of connective tissue, en route to catabolism in the liver or excretion in the urine. Moreover, it was shown that chondroitin sulfate i.p. injected to rats is excreted in the urine, part as polymeric chondroitin sulfate and part as low-molecular-weight degradation products.¹⁸ Heparan sulfate is present in almost all mammalian tissues, but since it is the main kidney glycosaminoglycan, part of the urinary heparan sulfate could be originated from the kidney; but cystectomy in rats did not produce detectable changes in excreted glycosaminoglycans,¹⁹ indicating that most of the urinary glycosaminoglycans does not come from the urinary tract. Thus, the changes observed in the urinary excretion of these compounds could be due to changes either in their structure and metabolism in tissues or in the renal permselectivity to these molecules. Since cartilage is the animal tissue that contains, by far, the highest chondroitin sulfate and keratan sulfate concentrations, it is likely that these urinary glycosaminoglycans, at least in part, come from the cartilage. If this is true, the analysis of urinary glycosaminoglycans could be a useful instrument for the early diagnosis and follow-up of joint diseases. The aim of the current study was to test this hypothesis in horses.

Although changes in urinary excretion of glycosaminoglycans with age have been studied in humans,⁹ there is little information concerning the age-related changes in other species. The horse is an accessible species with considerable longevity and is a good candidate for this non-human investigation. Furthermore, joint pain and loss of mobility are common causes of poor performance and early retirement of equine athletes.²⁰ It is important to understand the changes that occur with age and sport training upon the urinary excretion of glycosaminoglycans to avoid confusion with the processes that occur in osteoarthritis.

Osteoarthritis, also referred to as degenerative joint disease, is a group of disorders characterized by alterations in the metabolism of articular cartilage that leads to physical degeneration of the cartilage accompanied by changes in the bones and soft tissues of affected joints.²¹ Development of osteoarthritis in horses is often associated with the stresses of racing and training,²² supporting the idea that mechanical stress is a relevant cause of osteoarthritis. Loss of articular cartilage and fibrillation are hallmarks of osteoarthritis, and it was shown that apoptosis is a major pathway of chondrocyte death in human osteoarthritis.23, 24

So the aim of the current study was to investigate if the urinary glycosaminoglycans, particularly chondroitin sulfate and keratan sulfate, reflect the changes that occur in cartilage with growth, sport activities, and osteoarthritis. We used an ion exchange chromatography-based procedure to isolate glycosaminoglycans from urine samples of sedentary and athletic horses of different genders and ages, either healthy or with osteoarthritis. These compounds were identified by agarose gel electrophoresis and degradation with Flavobacterium heparinum chondroitinase B, chondroitinase AC, and heparitinase II. The urinary excretion of keratan sulfate and the structure of the urinary chondroitin sulfate were also analyzed. Keratan sulfate and chondroitin 4-sulfate were also immunolocalized by confocal microscopy in horse articular cartilage.